Plasmodium falciparum is the most pathogenic species of human malaria. The asexual parasites of this species in red blood cells produce several proteins characterized by an unusually high content of the amino acid histidine. One of these proteins is designated Histidine Rich Protein II (HRP-II). HRP-II is synthesized throughout the asexual stage of the parasite and is released from infected cells into the blood stream.
The HRP-II gene has been isolated and sequenced. The predicted amino acid sequence includes 34% histidine, 37% alanine and 10% aspartic acid (Wellems and Howard. 1986. PNAS 83, 6065-6069). The HRP-II protein is also unusual in that it contains many tandem repeats of the amino acid sequence Ala-His-His and a related sequence comprising (Wellems and Howard) about 80% of the sequence of the protein. The functions of HRP-II and the other histidine-rich proteins are not yet known, but HRP-II is of particular interest for early detection of malarial infection because it is transported out of infected cells into the extracellular medium where it can be detected in the blood of infected patients. Highly sensitive immunoassays for detection of HRP-II are therefore desirable as a means for detecting malaria infection at an early stage of the disease.
Antibodies specific for HRP-II are known in the art. Monoclonal antibody MAb-87 and its subclones, raised to the intact HRP-II protein, have been described by Howard, et al. (1986) J. Cell Biol. 103, 1269-1277; Rock, et al. (1987) Parasitology 95, 209-227 and Panton, et al. (1989) Molec. Biochem. Parasitology 35, 149-160. MAb-87 and its subclones are also disclosed in WO 89/01785 to Taylor. However, Mab-87 and its related monoclonals are not suitable for diagnosis of malaria, as they are capable of detecting a minimum of only 0.05% parasitemia in an enzyme linked immunosorbent assay (ELISA). Suppl. to Amer. J. Trop. Med. Hyg. 1991. 45(3): 248-249. Polyclonal antibodies to a fragment of the HRP-II protein containing Ala-His-His and Ala-Ala-Asp repeats have been reported by Knapp, et al. (1988. Behring Inst. Mitt. 82, 349-359). Wellems, et al. (1987. Cell 49, 633-642) have produced polyclonal antisera to a synthetic peptide having a similar amino acid sequence and these antibodies bind to the HRP-II protein. Wellems and Howard disclose these same polyclonal antisera and synthetic peptide in U.S. Pat. No. 5,130,416. The foregoing authors and inventors also describe the use of the various anti-HRP-II antibodies to detect HRP-II antigen in immunoassays such as Western blots and ELISA's.